Melatonin derivatives and medicine containing same

ABSTRACT

The invention concerns melatonin derivatives of formula (I) wherein: R 1  represents H, a C 1 -C 4  alkyl group or a C 1 -C 4  alkoxy group; R 2  represents H or a C 1 -C 4  alkyl group; R 3  represents H, methyl or a halogen atom; R 4 , R 5  represent individually H or a halogen atom; R 6  represents H or a C 1 -C 4  alkyl group, or a pharmaceutically acceptable salt thereof. The invention also concerns a medicine comprising said derivatives.

[0001] The present invention relates to novel melatonin derivativescapable of releasing nitrogen monoxide (NO) and melatonin underphysiological conditions.

[0002] Another subject matter of the invention is these melatoninderivatives for their application as therapeutically active substances.

[0003] Nitrogen monoxide is a product of NO-synthetases in all animalorgans, where it plays varied roles in maintaining the physiologicalfunctions, but especially in pathological situations, where it issynthesized in much greater amounts. It is a vasodilator and anantiaggregant for platelets and leukocytes with other cell types. It isinvolved in nerve transmissions, neither cholinergic nor adrenergic, butalso in excitotoxicity processes of glutamate in the nervous system. Inneurodegenerative processes, it is still difficult to take intoconsideration the involvement of NO and/or of oxygen derivatives.According to several authors, NO would rather have a protective rolewith regard to the neurons and would have an antioxidizing role bycombining with oxygenated radicals. However, when it binds tosuperoxide, it generates peroxynitrite, which, in turn, modifiesmacromolecules and very particularly DNA. The fragmentation of DNAconstitutes a phase in the apoptosis process.

[0004] As NO is an excellent scavenger of superoxide anions with thegeneration of peroxynitrite, protection of tissues can only be ensuredin the presence of compounds capable of scavenging peroxynitrite, suchas melatonin.

[0005] Melatonin or N-acetyl-5-methoxytryptamine is a hormonesynthesized in the pineal gland and in the retina, especially during thenight and in young animals. Its level decreases with age and during thedevelopment of Alzheimer's disease. It controls the physiologicalfunctions associated with the circadian rhythm and is involved in theimmune response.

[0006] It has recently been proposed that its essential role is toscavenge radicals and strong oxidants. These interactions do not requirerecognition with specific receptors but are involved in thephysiopathological processes of the living world. This hypothesis isbased on direct experimental results but also on the effects ofmelatonin in protecting proteins, membrane lipids and DNA from oxidativedamage due to radical processes. In particular, melatonin generallyprevents lipid peroxidation, both in vitro and in vivo.

[0007] The hydroxyl radical is the major toxic entity originating fromoxygen in living organisms. Its level increases with age. It isresponsible for the initiation of radical chain reactions whichcharacterize oxidative stress. There exists no specific enzyme systemcapable of inactivating it, as is the case for the superoxide anion.This is the role of antioxidants (vitamins C and E, glutathione,carotene, and the like). Melatonin is among the most effective of themsince it reacts with this radical with a rate close to the diffusionlimit. On the other hand, it does not react or reacts very slowly withthe superoxide anion.

[0008] It is also involved in the redox balance, maintaining thehomeostasis of glutathione and initiating the synthesis of genes ofantioxidant enzymes.

[0009] Its properties make it possible to explain why melatonin reducesthe toxicity of glutamate in neuronal cultures and the undesirableeffects during anticancer chemotherapies. In this case, thesetherapeutic effects are additional to its oncostatic properties observedwith respect to cultures of human cancer cells and with respect tocertain cancers.

[0010] Finally, recent results seem encouraging for the use of melatoninin the prophylaxis of the appearance of gastric ulcers and of migraine.

[0011] Among the NO donors currently known, only diazenium-diolates,generally known as NONOates, spontaneously and quantitatively releasetheir nitrosyl groups as NO.

[0012] The other NO donors release NO after reductive or oxidativestages. The only ones used as vasodilating medicaments in cardiovasculardiseases, organic nitrates and sydnonimines, exhibit complex in vitroand poorly known in vivo decomposition mechanisms. Organic nitrates aremetabolized in the walls of the blood vessels. Molsidomine orpirsidomine are hydrolyzed enzymatically in the liver and release activecompounds which spontaneously generate NO and the superoxide anion, invitro.

[0013] However, none of these compounds simultaneously releases anantioxidant capable in particular of scavenging peroxynitrite andnitrogen monoxide. Furthermore, melatonin derivatives used asmedicaments are known from patent applications EP-A-513 702 and EP-A-543659.

[0014] It is thus desirable to prepare compounds capable ofsimultaneously releasing both antioxidants: nitrogen monoxide and anantioxidant capable in particular of scavenging peroxynitrite.Furthermore, such compounds must exhibit a sufficient lipophilic natureto be physiologically acceptable, in particular for crossing thehematoencephalic barrier.

[0015] The compounds which are a subject matter of the present inventionsimultaneously solve the abovementioned problems.

[0016] According to the invention, the melatonin derivative correspondsto the formula:

[0017] in which:

[0018] R₁ represents H, a C₁ to C₄ alkyl group or a C₁ to C₄ alkoxygroup,

[0019] R₂ represents H or a C₁ to C₄ alkyl group,

[0020] R₃ represents H, methyl or a halogen atom,

[0021] R₄ and R₅ individually represent H or a halogen atom,

[0022] R₆ represents H or a C₁-C₄ alkyl group,

[0023] or a pharmaceutically acceptable salt of the latter.

[0024] Preferably, R₂=H, R₆=CH₃ and R₁=CH₃.

[0025] According to a preferred alternative form of the invention, themelatonin derivative corresponds to the formula:

N1-nitrosomelatonin (Niomel)

[0026] Another subject matter of the invention is the compounds offormula I or II for their application as therapeutically activesubstances.

[0027] Another subject matter of the invention is a pharmaceuticalcomposition comprising a compound of formula I or II and apharmacologically acceptable excipient.

[0028] Mention is made, among the therapeutic applications, ofcardiovascular diseases, ischemia reperfusion injuries, parasiticdiseases, inflammatory diseases, neuro-degenerative diseases, inparticular Alzheimer's disease, myopathies or falciform anemias.

[0029] Another subject matter of the invention is a process for thepreparation of the melatonin derivatives of formula I, characterized inthat a precursor of formula:

[0030] in which R₁ to R₆ have the same meanings as those indicated forthe formula I, is brought into contact, in protic organic solution, withan aqueous sodium nitrite solution and then the mixture is basified, soas to obtain a derivative according to the invention.

[0031] The melatonin derivative of formula II is prepared in thefollowing way:

[0032] An aqueous sodium nitrite solution is added to a solution, cooledto 4° C., of melatonin in acetic acid and methanol. After basifying witha saturated sodium bicarbonate solution, the N1-nitrosomelatonin isextracted with dichloromethane and the organic solution is dried withmagnesium sulfate and then evaporated under reduced pressure. Theresidue is taken up in ethyl acetate and hexane until crystals areobtained. The crystals are recovered by filtration, dried and stored atambient temperature with the exclusion of light.

[0033] This compound exhibits a melting point of: 138° C.

[0034] Chromatographic analysis on a Hypersil C18 column, elution usinga gradient of 10 to 50% of acetonitrile and 0.05% of trifluoroaceticacid over 50 minutes, 215 nm detection, results in a peak at 431,whereas the melatonin peak is at 258.

[0035] The characteristic band of the light absorption spectrum of theproduct in phosphate solution from pH 4 to pH 9 is situated at 346 nm.

[0036] A significant peak (m/z M+H⁺=262) is observed by “fast atomicbombardment” mass spectrometry.

[0037] The elemental analysis for carbon, proton and nitrogencorresponds to the molar mass 261 and to the formula

C₁₃H₁₅N₃O₃.

Biological Activity

[0038] The biological activity of the derivative of formula I wascompared with dipropylenetriamine NONOate (A), melatonin (B) and adipropylenetriamine NONOate and melatonin (1/2) mixture (C) on the cellpreparation of the cell proliferation of the P 815 line.

[0039] The cells are incubated with the products in RPMI medium withphenol red for 7 hours. Tritiated thymidine is added after incubatingfor 3 hours.

[0040] The results are shown on the curve in the single appended figure,where the concentration of product (μM) is carried on the abscissa andthe radioactivity associated with the DNA (cpm) is carried on theordinate.

[0041] The IC₅₀ for the compound of formula I occurs at a concentrationof 280 μM; the IC₅₀ for dipropylene-triamine NONOate (A) is 150 μM.

[0042] It is thus found that the melatonin derivative according to theinvention displays an activity comparable with that ofdipropylenetriamine NONOate.

1. Melatonin derivative of formula:

in which: R₁ represents H, a C₁ to C₄ alkyl group or a C₁ to C₄ alkoxygroup, R₂ represents H or a C₁ to C₄ alkyl group, R₃ represents H,methyl or a halogen atom, R₄ and R₅ individually represent H or ahalogen atom, R₆ represents H or a C₁-C₄ alkyl group, or apharmaceutically acceptable salt of the latter.
 2. Melatonin derivativeaccording to claim 1, characterized in that R₂=H, R₆=CH₃ and R₁=CH₃. 3.Melatonin derivative according to claim 2 of formula:

N1-nitrosomelatonin (Niomel)
 4. Melatonin derivative according to one ofclaims 1 to 3, for its application as therapeutically active substance.5. Melatonin derivative according to one of claims 1 to 3, for itsapplication as therapeutically active substance in the treatment ofcardiovascular diseases, ischemia reperfusion injuries, parasiticdiseases, inflammatory diseases, neurodegenerative diseases, inparticular Alzheimer's disease, myopathies or falciform anemias. 6.Pharmaceutical composition, characterized in that it comprises amelatonin derivative of formula I and an active pharmaceuticalexcipient.
 7. Process for the preparation of a melatonin derivativeaccording to claim 1, characterized in that a precursor of formula:

in which R₁ to R₆ have the same meanings as in claim 1, is brought intocontact, in protic organic solution, with an aqueous sodium nitritesolution and then the mixture is basified, so as to obtain a derivativeaccording to one of claims 1 to 3.